In the fields of clinical examinations, food analyses etc., amino acid analysis is of ever-increasing necessity, and demands for rapidity, simplicity and high reliability have been growing accordingly. Under such circumstances, bioassays utilizing enzyme reactions have attracted attention in the art because of substrate specificity of the enzymes and speed of reaction. Enzymes have taken a major role in various methods of amino acid analysis. In the clinical field, because of limitations of sample size and analysis time, there has been a need to develop a biosensor for amino acid determination which achieves not only improved rapidity and simplicity but also economy by reducing the need for costly reagents. In the food industry, too, a biosensor for amino acid determination excellent in rapidity, simplicity and economy has been demanded for process control, quality control and also for taste analysis as certain amino acids such as glutamic acid and alanine are substances with desirable tastes.
Known biosensors for amino acid determination include (1) an enzyme electrode comprising an amino-acid oxidase immobilized on an oxygen electrode or a hydrogen peroxide electrode, as disclosed in Analytical Chemistry, 47, 1359 (1975); (2) an analyzer comprising an amino-acid oxidase combined with a mass analyzer for improving rapidity, as disclosed in JP-A-60-52765 (the term "JP-A" as used herein means an "unexamined published Japanese patent application"); and (3) an enzyme electrode comprising an amino-acid oxidase and an amino-acid dehydrogenase or an aminotransferase immobilized on an electrode for increasing sensitivity, as disclosed in JP-A-60-73354.
Attempts were also made on (4) an enzyme electrode for alanine determination using glutamate-pyruvate transaminase and pyruvate oxidase immobilized on an electrode, as disclosed in JP-A-62-162952 and (5) an enzyme electrode using glutamatepyruvate transaminase and glutamate oxidase, as disclosed in Chemistry Express, Vol. 5, p. 125 (1990).
Because all the above described enzyme electrodes (1) to (5) use an oxidase, they are, in principle, susceptible to influences of dissolved oxygen or reducing substances. With respect to rapidity, only electrode (2) has a rate of response of within 1 minute. With respect to sensitivity, only electrodes (3) and (5) have a detection concentration limit on the order of .mu.M or less. Thus, an enzyme electrode for practical use which satisfies rapidity, high sensitivity and substantially sufficient durability has not yet been developed.